1. Technical Field of the Invention
The present invention relates to a method for producing an L-amino acid, and a bacterium used therefor. More precisely, the present invention relates to a methane-utilizing bacterium having improved L-amino acid producing ability and a method for producing an L-amino acid utilizing the bacterium.
2. Description of the Related Art
Conventionally, L-amino acids such as L-lysine, L-glutamic acid, L-threonine, L-leucine, L-isoleucine, L-valine and L-phenylalanine are produced by fermentation utilizing coryneform bacteria belonging to the genus Brevibacterium, Corynebacterium or Microbacterium (Amino Acid Fermentation, the Japan Scientific Societies Press [Gakkai Shuppan Center], pp. 195–215, 1986). Furthermore, microorganisms of the genus Bacillus, Streptomyces, Penicillium (U.S. Pat. No. 3,220,929), Pseudomonas, Arthrobacter, Serratia, Aerobacter, Candida (U.S. Pat. No. 3,563,857), Escherichia (Japanese Patent Laid-open (Kokai) No. 5-244970) and the like can also be utilized in the production of L-amino acids.
To improve productivity of these microorganisms, bacterial strains isolated from nature, or artificial mutants of the bacterial strains, have been used. Furthermore, various techniques have been disclosed for increasing L-amino acid producing ability by enhancing L-amino acid biosynthesis enzymes using recombinant DNA techniques (U.S. Pat. Nos. 4,278,765, 4,346,170 and 6,040,160).
Methanol is a raw material often used in fermentation which is inexpensive and widely and easily available. Methods for producing L-amino acids by fermentation of methanol have been known using microorganisms that belong to the genus Achromobacter or Pseudomonas (Japanese Patent Publication (Kokoku) No. 45-25273), Protaminobacter (Japanese Patent Laid-open Publication (Kokai) No. 49-125590), Protaminobacter or Methanomonas (Japanese Patent Laid-open (Kokai) No. 50-25790), Microcyclus (Japanese Patent Laid-open (Kokai) No. 52-18886), Methylobacillus (Japanese Patent Laid-open (Kokai) No. 4-91793), Bacillus (Japanese translation of PCT international application Patent (Kohyo) No. 3-505284 (WO90/12105)) and the like. The inventors of the present invention to date have developed methods for producing L-amino acids using Methylophilus bacteria employing breeding techniques utilizing artificial mutagenesis and recombinant DNA (WO00/61723).
Techniques are also known for enhancing L-amino acid producing ability by introducing genes coding for glycolytic enzymes such as glucose-6-phosphate isomerase (International Patent Publication No. 01/02542 (WO 01/02542 A1)), fructose phosphotransferase (International Patent Publication No. 01/48146 (WO 01/48146 A1)) and enolase (International Patent Publication No. 01/02543 (WO 01/02543 A1)).
Many methanol-utilizing bacteria including enterobacteria have the Entner-Doudoroff pathway as one of their methanol metabolic pathways. This pathway involves 6-phosphogluconate dehydratase (abbreviated as “EDD” hereinafter), which catalyzes a reaction to produce 2-keto-3-deoxy-6-phosphogluconate from 6-phosphogluconic acid, and 2-keto-3-deoxy-6-phosphogluconate aldolase (abbreviated as “EDA” hereinafter), which cleaves 2-keto-3-deoxy-6-phosphogluconate to produce glyceraldehyde-3-phosphate and pyruvic acid. Genes coding for EDD and EDA have been cloned from Escherichia coli, Zymomonas mobilis and so forth, and their nucleotide sequences have been reported (Mol. Microbiol. 5, 2901–2911; J. Bacteriol. 172 (12), 7227–7240 (1990)). The nucleotide sequences of the gene coding for EDD (edd) and the gene coding for EDA (eda) of Escherichia coli are registered as GenBank accession number L20897. Furthermore, the nucleotide sequence of the eda gene of Zymomonas mobilis is registered as GenBank accession number X58364, and the nucleotide sequence of the edd gene is registered as GenBank accession number M60615 M37982 in the database.
There is clearly a need in the art for efficient, low-cost, productive methods of obtaining amino acids for both agricultural and nutritional uses. The relationship between the Entner-Doudoroff pathway and the productivity of L-amino acids has not previously been described. The present invention describes this relationship and a method of exploiting it.